The Principal Investigator's long term research interest is the evolution of innate immune mechanisms, particularly the role of complement as a mediator of innate immunity and a modulator of adaptive immunity. Comparative studies on shark complement, at the protein, gene and functional level, is the focus of this application.The shark represents a stage in complement evolution where for the first time the complement system is functioning in an environment that also has a functional adaptive immune system. Furthermore, the shark is also the most primitive vertebrate to possess complement derived iytic activity. The goal of this study is to provide information on the biochemical, structural, genetic and functional changes that have occurred in components and which likely have arisen (a) to form the activation cascades and the terminal lytic pathway, and (b) to permit components to specifically interact with components of the adaptive system, a functional role not required of complement in primitive organisms lacking adaptive immune mechanisms. The specific aims are: (1) to functionally characterize the serum proteins encoded by the two Bf/C2 genes (gcBf-1 and gcBf-2), (2) to isolate and biochemically characterize the protein encoded by the second C3 gene (gcC3-2), (3) to characterize the complement derived anaphylatoxins, C3a, C4a and putative C5a, (4) to complete the characterization of putative shark factor H and define its role as a cofactor for factor I cleavage activity, (5) to examine the glycosylation pattern of factor I isoforms, and (6) to predict the molecular composition of shark Membrane Attack Complex (MAC). Research methods will include expression of recombinant proteins in a prokaryotic system, protein isolation and purification, ion exchange and affinity chromatography, protein and nucleic acid sequencing, analysis of protein glycosylation, gene cloning, Westem blotting, miscellaneous functional assays such as chemotaxis and anaphylaxis, and the development of shark-specific reagents and protocols. The study will attempt to answer the questions: what evolutionary changes have occurred in the shark system that make it different from that found in lower vertebrates which lack components of an adaptive immune system, and is shark MAC assembled from components homologous to those found in mammalian MAC, i.e., C5 through C97 Information on anaphylatoxins could, potentially, be of medical interest and application.